Elucidating the molecular mechanisms of sepsis: Identifying key aging-related biomarkers and potential therapeutic targets in the treatment of sepsis.

BACKGROUND: Sepsis remains a crucial global health issue characterized by high mortality rates and a lack of specific treatments. This study aimed to elucidate the molecular mechanisms underlying sepsis and to identify potential therapeutic targets and compounds. METHODS: High-throughput sequencing data from the GEO database (GSE26440 as the training set and GSE13904 and GSE32707 as the validation sets), weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, alongside a combination of PPI and machine learning methods (LASSO and SVM) were utilized. RESULTS: WGCNA identified the black module as positively correlated, and the green module as negatively correlated with sepsis. Further intersections of these module genes with age-related genes yielded 57 sepsis-related genes. GO and KEGG pathway enrichment analysis, PPI, LASSO, and SVM selected six hub aging-related genes: BCL6, FOS, ETS1, ETS2, MAPK14, and MYC. A diagnostic model was constructed based on these six core genes, presenting commendable performance in both the training and validation sets. Notably, ETS1 demonstrated significant differential expression between mild and severe sepsis, indicating its potential as a biomarker of severity. Furthermore, immune infiltration analysis of these six core genes revealed their correlation with most immune cells and immune-related pathways. Additionally, compounds were identified in the traditional Chinese medicine Danshen, which upon further analysis, revealed 354 potential target proteins. GO and KEGG enrichment analysis of these targets indicated a primary enrichment in inflammation and immune-related pathways. A Venn diagram intersects these target proteins, and our aforementioned six core genes yielded three common genes, suggesting the potential efficacy of Danshen in sepsis treatment through these genes. CONCLUSIONS: This study highlights the pivotal roles of age-related genes in the molecular mechanisms of sepsis, offers potential biomarkers, and identifies promising therapeutic compounds, laying a robust foundation for future studies on the treatment of sepsis.

A Novel Based-Network Strategy to Identify Phytochemicals from Radix Salviae Miltiorrhizae (Danshen) for Treating Alzheimer's Disease.

Alzheimer's disease (AD) is a common age-related neurodegenerative disease that strikes millions worldwide. Herein, we demonstrate a new approach based on network target to identify anti-AD compounds from Danshen. Network pharmacology and molecular docking were employed to establish the DS-AD network, which mainly involved apoptosis of neuron cells. Then network scoring was confirmed via Connectivity Map analysis. M308 (Danshenxinkun D) was an anti-AD candidate with a high score (p < 0.01). Furthermore, we conducted ex vivo experiments with H2O2-treated PC12 cells to verify the neuroprotective effect of Salvia miltiorrhiza-containing plasma (SMP), and UPLC-Q-TOF/MS and RT-qPCR were performed to demonstrate the anti-AD activity of M308 from SMP. Results revealed that SMP could enhance cell viability and level of acetylcholine. AO/EB staining and Mitochondrial membrane potential (MMP) analysis showed that SMP significantly suppressed apoptosis, which may be due to anti-oxidative stress activity. Moreover, the effects of M308 and SMP on expressions of PSEN1, DRD2, and APP mRNA were consistent, and M308 can significantly reverse the expression of PSEN1 and DRD2 mRNA in H2O2-treated PC12 cells. The strategy based on the network could be employed to identify anti-AD compounds from Chinese herbs. Notably, M308 stands out as a promising anti-AD candidate for development.

Radix Salviae miltiorrhizae improves bone microstructure and strength through Wnt/ß-catenin and osteoprotegerin/receptor activator for nuclear factor-κB ligand/cathepsin K signaling in ovariectomized rats.

Although radix Salviae miltiorrhizae (RSM) is reported to exhibit the antiosteoporotic effect in preclinical study, the underlying mechanism is unclear. To this end, ovariectomized (OVX) rats were employed with administration of RSM (5 g/kg) for 14 weeks. The disturbed serum levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), tartrate-resistant acid phosphatase, and receptor activator of nuclear factor-κB ligand (RANKL) in OVX rats were improved by RSM treatment. Furthermore, supplement of RSM to OVX rats resulted in an increase in femoral bone mineral density and bone strength as well as an improvement in bone microstructures. Moreover, the decreased expression of phosphor (p)-LRP6, insulin-like growth factor-1(IGF-1), ALP, and OPG, as well as increased expression of RANKL and cathepsin K in the tibias and femurs of OVX rats were shifted by RSM treatment. Additionally, RSM reversed the decreased ratio of p-glycogen synthase kinase 3ß (GSK3ß) to GSK3ß and increased ratio of p-ß-catenin to ß-catenin in OVX rats. Altogether, it is suggestive that RSM improves bone quantity and quality by favoring Wnt/ß-catenin and OPG/RANKL/cathepsin K signaling pathways in OVX rats thereby suggesting the potential of this herb to be a novel source of antiosteoporosis drugs.

A Biomedical Investigation of the Hepatoprotective Effect of Radix salviae miltiorrhizae and Network Pharmacology-Based Prediction of the Active Compounds and Molecular Targets.

Radix salviae miltiorrhizae (Danshen in Chinese), a classic traditional Chinese medicine (TCM) herb, has been used for centuries to treat liver diseases. In this study, the preventive and curative potential of Danshen aqueous extract on acute/chronic alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) was studied. The in vivo results indicated that Danshen could alleviate hepatic inflammation, fatty degeneration, and haptic fibrogenesis in ALD and NAFLD models. In the aspect of mechanism of action, the significant reduction in MDA levels in both ALD and NAFLD models implies the decreased levels of oxidative stress by Danshen. However, Danshen treatment could not activate the internal enzymatic antioxidant system in ALD and NAFLD models. To further explore the hepatoprotective mechanism of Danshen, an in silico-based network pharmacology approach was employed in the present study. The pharmacological network analysis result revealed that six potential active ingredients such as tanshinone iia, salvianolic acid b, and Danshensu may contribute to the hepatoprotective effects of Danshen on ALD and NAFLD. The action mechanism may relate with regulating the intracellular molecular targets such as PPARα, CYP1A2, and MMP2 for regulation of lipid metabolism, antioxidant and anti-fibrogenesis by these potential active ingredients. Our studies suggest that the combination of network pharmacology strategy with in vivo experimental study may provide a forceful tool for exploring the mechanism of action of traditional Chinese medicine (TCM) herb and developing novel bioactive ingredients.

Effects of Shenlian extract on experimental atherosclerosis in ApoE-deficient mice based on ultrasound biomicroscopy.

BACKGROUND: This study directly and dynamically investigated the effects of SL extract (i.e., a combination of Radix Salviae miltiorrhizae and Andrographis paniculata extract) on plaque progression in vivo by high resolution ultrasound biomicroscopy (UBM). METHODS: An atherosclerosis model was established by placing a perivascular collar on the right common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice. Thickness, plaque area and local blood flow were observed by UBM, pathological changes were observed by histochemical staining, and lipid levels were measured by respective commercially available kits. RESULTS: Compared with the model group, the SL extract groups showed reduced wall thickness of the aortic arch (GC: P = 0.001, P = 0.002, and P < 0.001; LC: P < 0.001, P < 0.001, and P < 0.001; BC: P = 0.027, P = 0.017, and P = 0.003; respectively), which presented with retarded plaque progression of the cartoid artery with concordantly increased blood flow (P = 0.002 and P < 0.001) as visualized in vivo by UBM. Histological analysis confirmed the reduction of carotid atherosclerosis. CONCLUSIONS: The SL extract inhibited the formation of atherosclerotic plaques in an ApoE-/- mice model by UBM analysis, and did so by effects that ameliorated local blood flow and improved blood lipid levels.

Deep Eutectic Solvent-Based Microwave-Assisted Method for Extraction of Hydrophilic and Hydrophobic Components from Radix Salviae miltiorrhizae.

Deep eutectic solvents (DESs) have attracted significant attention as a promising green media. In this work, twenty-five kinds of benign choline chloride-based DESs with microwave-assisted methods were applied to quickly extract active components from Radix Salviae miltiorrhizae. The extraction factors, including temperature, time, power of microwave, and solid/liquid ratio, were investigated systematically by response surface methodology. The hydrophilic and hydrophobic ingredients were extracted simultaneously under the optimized conditions: 20 vol% of water in choline chloride/1,2-propanediol (1:1, molar ratio) as solvent, microwave power of 800 W, temperature at 70 °C, time at 11.11 min, and solid/liquid ratio of 0.007 g·mL-1. The extraction yield was comparable to, or even better than, conventional methods with organic solvents. The microstructure alteration of samples before and after extraction was also investigated. The method validation was tested as the linearity of analytes (r² > 0.9997 over two orders of magnitude), precision (intra-day relative standard deviation (RSD) < 2.49 and inter-day RSD < 2.96), and accuracy (recoveries ranging from 95.04% to 99.93%). The proposed DESs combined with the microwave-assisted method provided a prominent advantage for fast and efficient extraction of active components, and DESs could be extended as solvents to extract and analyze complex environmental and pharmaceutical samples.

Systematic investigation of Radix Salviae for treating diabetic peripheral neuropathy disease based on network Pharmacology.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a debilitating complication of diabetes mellitus with limited available treatment options. Radix Salviae, a traditional Chinese herb, has shown promise in treating DPN, but its therapeutic mech-anisms have not been systematically investigated. AIM: Radix Salviae (Danshen in pinin), a traditional Chinese medicine (TCM), is widely used to treat DPN in China. However, the mechanism through which Radix Salviae treats DPN remains unclear. Therefore, we aimed to explore the mechanism of action of Radix Salviae against DPN using network pharmacology. METHODS: The active ingredients and target genes of Radix Salviae were screened using the TCM pharmacology database and analysis platform. The genes associated with DPN were obtained from the Gene Cards and OMIM databases, a drug-com-position-target-disease network was constructed, and a protein-protein inter-action network was subsequently constructed to screen the main targets. Gene Ontology (GO) functional annotation and pathway enrichment analysis were performed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) using Bioconductor. RESULTS: A total of 56 effective components, 108 targets and 4581 DPN-related target genes of Radix Salviae were screened. Intervention with Radix Salviae for DPN mainly involved 81 target genes. The top 30 major targets were selected for enrichment analysis of GO and KEGG pathways. CONCLUSION: These results suggested that Radix Salviae could treat DPN by regulating the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway. Therefore, Danshen may affect DPN by regulating inflammation and apoptosis.

Study on therapeutic mechanism of total salvianolic acids against myocardial ischemia-reperfusion injury based on network pharmacology, molecular docking, and experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salviae miltiorrhizae, also known as Danshen in Chinese, effectively activates the blood and resolves stasis. Total salvianolic acids (SA) is the main active ingredient of Danshen, and related preparations, such as salvianolate injection are commonly used clinically to treat myocardial ischemia-reperfusion injury (MIRI). However, the potential targets and key active ingredients of SA have not been sufficiently investigated. AIM OF THE STUDY: This study aimed to investigate the mechanism of action of SA in treating MIRI. MATERIALS AND METHODS: Network pharmacology and molecular docking techniques were used to predict SA targets against MIRI. The key acting pathway of SA were validated by performing experiments in a rat MIRI model. RESULTS: Twenty potential ingredients and 54 targets of SA in treating MIRI were identified. Ingredient-target-pathway network analysis revealed that salvianolic acid B and rosmarinic acid had the highest degree value. Pathway enrichment analysis showed that SA may regulate MIRI through the IL-17 signaling pathway, and this result was confirmed in the rat MIRI experiment. CONCLUSION: The results of this study indicate that SA may protect MIRI by regulating the IL-17 pathway.

Tanshinol suppresses osteosarcoma by specifically inducing apoptosis of U2-OS cells through p53-mediated mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salviae miltiorrhizae (also called Danshen in traditional Chinese medicine) is a famous herbal medicine, which has been frequently used to treat blood stasis syndrome including osteosarcoma (OS) in traditional Chinese medicine. Main components of Danshen have been assumed to exhibit anti-OS capacity. Nevertheless, tanshinol (TS, main component of Danshen)'s efficacy and mechanism in OS hasn't been clearly described ever since. This drew our attention, since OS is the most frequent primary bone carcinomas in children and adolescents, with a high incidence and fatality rate. Unfortunately, chemotherapy for OS has faced many clinical challenges due to the increasing chemoresistance and recurrence. This study was then designed to deeply explore TS's role in OS therapy. AIM OF THE STUDY: To explore the anti-OS efficacy and mechanism of TS, we conducted in vivo and in vitro experiments by using a zebrafish xenograft model and U2-OS cells. MATERIALS AND METHODS: CCK-8 assay, DAPI and γ-H2A.X immunofluorescence staining, and flow cytometry (apoptosis verification) were employed to determine the anti-proliferative and pro-apoptotic effects of TS. qPCR and Western blot were used to examine TS's molecular actions and mechanism on apoptosis of U2-OS cells. RESULTS: The in vivo data showed that TS significantly inhibited U2-OS tumor growth in larval zebrafish from 2 to 20 ng/mL. In vitro data indicated that TS exerted significant anti-proliferative and pro-apoptotic effects on U2-OS cells in a dose-dependent manner. Moreover, TS has no inhibitory effect on bMSCs, suggesting its safety on normal bone-forming cells. Molecular data illustrated that TS obviously activated the p53 signaling-related proteins (p-p53, Bax, CASP3, CASP9) and its upstream JNK (p-JNK, p-c-JUN) and ATM (p-ATM) signaling molecules through phosphorylation and cleavage, followed by up-regulation of the pro-apoptotic genes, NOXA, PUMA, TP53, BAX, and BIM, and down-regulation of Bcl-2 protein. CONCLUSION: In sum, TS specifically induced apoptosis of U2-OS cells by activating p53 signaling pathways, indicating TS as a promising candidate for OS treatment.

Metabolomics study on the intervention effect of Radix Salviae Miltiorrhizae extract in exercise-induced exhaustion rat using gas chromatography coupled to mass spectrometry.

The metabolomics approach based on the gas chromatography coupled to mass spectrometry (GC-MS) was adopted to explore the underlying mechanism of the anti-fatigue effect of Radix Salviae Miltiorrhizae (RSM), a famous herbal medicine in China used for multiple biological functions, in load-weighted swimming test in rat, combined with biochemical parameters evaluations. As a result, the metabolomics study followed by orthogonal partial least-square (OPLS) analysis could differentiate metabolic profiling between the control and exhaustive exercise group, showing the rats underwent an obvious metabolic perturbation, whereas RSM treatment restored scores plot close to normal and showed regulatory effects on the muscle metabolic profiles. The changed metabolic pathways of the potential biomarkers in response to the effect of RSM treatment for exhaustive exercise rats included in glucose metabolism (glucose, lactic acid, alanine), glutathione metabolism (glycine, glutamate, 5-oxo-proline), TCA cycle (succinic acid), arginine biosynthesis (glutamine, ornithine, urea), glyoxylate and dicarboxylate metabolism (serine, glycine), oxidative stress (taurine) and purine metabolism (inosine). In addition, intervention of RSM increased hepatic glycogen, muscle glycogen and serum glucose, and decreased triglyceride and blood urea nitrogen levels, indicating RSM treatment may regulate energy metabolism by increasing the rate of fat utilization, decrease the protein and carbohydrate utilization. Furthermore, RSM reduced exhaustive exercise-induced accumulation of the lipid peroxidation byproduct malonaldehyde and elevated antioxidants' levels, including reduced glutathione and superoxide dismutase, which might be a positive reflection of improved oxidant-antioxidant balance. Moreover, RSM could protect against exercise-induced muscle damage by attenuating creatine kinase release. In summary, RSM provided a good anti-fatigue effect by regulating energy metabolism, oxidant-antioxidant balance, and the endogenous metabolites in the exercising muscle. This study demonstrates that metabolomics is an effective tool for the estimation of the potential anti-fatigue effect of RSM and for the illustration of its pharmacological mechanism.

Exploring the Pharmacological Mechanism of Radix Salvia Miltiorrhizae in the Treatment of Radiation Pneumonia by Using Network Pharmacology.

BACKGROUND: Radiation pneumonia (RP) is the most common complication of radiotherapy to the thorax and seriously affects the survival rate and quality of life of patients. Radix Salviae Miltiorrhizae (RSM) is an ancient Chinese medicine, whose main pharmacological effect is to promote blood circulation and remove stasis. A growing number of studies have proved that RSM has a good effect on RP. However, the underlying mechanism is still unclear and needs to be fully elucidated. METHODS: The effective components and predictive targets of RSM were analyzed by Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and the related targets of RP were predicted by GeneCards database. The common targets of the two targets mentioned above were analyzed by protein-protein interaction on the STRING website, GO and KEGG analysis on the DAVID website, visualization by CytoScape3.7.0, and screening for Hubber gene by cytoHubber plug-in. RESULTS: A search of the TCMSP database revealed that RSM contains 65 chemical constituents and 165 potential protein targets. A total of 2,162 protein targets were found to be associated with RP. The top 10 hub genes were obtained by MCC algorithm for 70 common genes, including TP53, CASP3, MAPK1, JUN, VEGFA, STAT3, PTGS2, IL6, AKT1, and FOS. By analyzing the Gene Ontology, The anti-radiation pneumonia effect of RSM is that it performs molecular functions (protein homodimerization activity) in the nucleus through three biological processes (positive regulation of transcription from RNA polymerase II promoter,Extrinsic apoptotic signaling pathway in absence of ligand and lipopolysaccharide-mediated signaling pathway). Through KEGG analysis, the mechanism of RSM treatment of radiation pneumonia may be through PI3K-Akt, HIF-1, TNF signaling pathways. CONCLUSIONS: Through network pharmacology analysis, we found the possible target genes of RSM on RP and revealed the most likely signaling pathway, providing theoretical basis for further elucidating the potential mechanism of RSM on RP.

Establishment of thrombin affinity column (TAC)-HPLC-MS/MS method for screening direct thrombin inhibitors from Radix Salviae Miltiorrhiae.

In order to develop an affinity HPLC method for screening direct thrombin inhibitors from Traditional Chinese Medicine (TCM), thrombin was immobilized on the glutaraldehyde-modified amino silica gel and was used as thrombin stationary phase. A thrombin affinity column (TAC) was made by packing the thrombin stationary phase into a bare column (2.0 * 1.0 mm, i.d.). The direct thrombin inhibitors could be screened through this TAC column. For the purpose of improvement of the discovery efficiency, a TAC-HPLC-MS/MS system was used to screen thrombin inhibitors from Radix Salviae Miltiorrhiae (RSM), a famous traditional Chinese medicine. After optimization of all the conditions, cryptotanshinone (Cry), dihydrotanshinone I (Dih-I) and tanshinone IIA (Tan-IIA) were screened out and identified as potential active components. The anticoagulant effects of these three compounds were tested by anticoagulant experiments in vitro. Furthermore, the interaction of three compounds with thrombin was studied by molecular docking. The result shows they have the potential to be used as preventive drugs. In short, this method can be used to screen anticoagulant drugs from traditional Chinese medicine, which provides convenience for screening anticoagulant drugs.

A network pharmacology-based study on the anti-hepatoma effect of Radix Salviae Miltiorrhizae.

BACKGROUND: Radix Salviae Miltiorrhizae (RSM), a well-known traditional Chinese medicine, has been shown to inhibit tumorigenesis in various human cancers. However, the anticancer effects of RSM on human hepatocellular carcinoma (HCC) and the underlying mechanisms of action remain to be fully elucidated. METHODS: In this study, we aimed to elucidate the underlying molecular mechanisms of RSM in the treatment of HCC using a network pharmacology approach. In vivo and in vitro experiments were also performed to validate the therapeutic effects of RSM on HCC. RESULTS: In total, 62 active compounds from RSM and 72 HCC-related targets were identified through network pharmacological analysis. RSM was found to play a critical role in HCC via multiple targets and pathways, especially the EGFR and PI3K/AKT signaling pathways. In addition, RSM was found to suppress HCC cell proliferation, and impair cancer cell migration and invasion in vitro. Flow cytometry analysis revealed that RSM induced cell cycle G2/M arrest and apoptosis, and western blot analysis showed that RSM up-regulated the expression of BAX and down-regulated the expression of Bcl-2 in MHCC97-H and HepG2 cells. Furthermore, RSM administration down-regulated the expression of EGFR, PI3K, and p-AKT proteins, whereas the total AKT level was not altered. Finally, the results of our in vivo experiments confirmed the therapeutic effects of RSM on HCC in nude mice. CONCLUSIONS: We provide an integrative network pharmacology approach, in combination with in vitro and in vivo experiments, to illustrate the underlying therapeutic mechanisms of RSM action on HCC.

Utilizing network pharmacology to explore the underlying mechanism of Radix Salviae in diabetic retinopathy.

INTRODUCTION: Radix Salviae (Dan-shen in pinyin), a classic Chinese herb, has been extensively used to treat diabetic retinopathy in clinical practice in China for many years. However, the pharmacological mechanisms of Radix Salviae remain vague. The aim of this study was to decrypt the underlying mechanisms of Radix Salviae in the treatment of diabetic retinopathy using a systems pharmacology approach. METHODS: A network pharmacology-based strategy was proposed to elucidate the underlying multi-component, multi-target, and multi-pathway mode of action of Radix Salviae against diabetic retinopathy. First, we collected putative targets of Radix Salviae based on the Traditional Chinese Medicine System Pharmacology database and a network of the interactions among the putative targets of Radix Salviae and known therapeutic targets of diabetic retinopathy was built. Then, two topological parameters, "degree" and "closeness certainty" were calculated to identify the major targets in the network. Furthermore, the major hubs were imported to the Database for Annotation, Visualization and Integrated Discovery to perform a pathway enrichment analysis. RESULTS: A total of 130 nodes, including 18 putative targets of Radix Salviae, were observed to be major hubs in terms of topological importance. The results of pathway enrichment analysis indicated that putative targets of Radix Salviae mostly participated in various pathways associated with angiogenesis, protein metabolism, inflammatory response, apoptosis, and cell proliferation. The putative targets of Radix Salviae (vascular endothelial growth factor, matrix metalloproteinases, plasminogen, insulin-like growth factor-1, and cyclooxygenase-2) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of Radix Salviae on diabetic retinopathy. CONCLUSIONS: Radix Salviae could alleviate diabetic retinopathy via the molecular mechanisms predicted by network pharmacology. This research demonstrates that the network pharmacology approach can be an effective tool to reveal the mechanisms of traditional Chinese medicine from a holistic perspective.

Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors.

Radix Salviae Miltiorrhiae (also known as DanShen (DS) in China), a popular herbal drug in traditional Chinese medicine (TCM) for promoting blood circulation and treating blood stasis, has been reported to possess potential anti-tumor effects. The aim of the study was to develop an effective and practical method for screening and identifying bioactive compounds from Radix Salviae Miltiorrhiae. In this work, the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 4 (FGFR4) dual-mixed/cell membrane chromatography (CMC) coupled with high performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrum (HPLC-ESI-IT-TOF-MSn) was established and successfully used to identify the active components from Radix Salviae Miltiorrhiae. Salvianolic acid C (SAC), tanshinone I (Tan-I), tanshinone IIA (Tan-IIA), and cryptotanshinone (C-Tan) were identified as bioactive components with EGFR and FGFR4 activities. MTT and kinase assay were performed to investigate inhibitory effects of these compounds against EGFR and FGFR4 cells growth in vitro. Both cell viability and kinase activity showed that cryptotanshinone acting on EGFR receptor and tanshinone IIA acting on FGFR4 receptor. In conclusion, the EGFR & FGFR4 dual-mixed/CMC can simultaneously screen the bioactive components from TCMs that act on both EGFR and FGFR4 receptors, which significantly improve the efficiency of specific bioactive components identification from a complex system.

Rapid separation and identification of multiple constituents in Danhong Injection by ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

To characterize and identify multiple constituents in Danhong injection (DHI), a fast ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF/MS) method was established and validated in the present study. A total of 63 compounds, including 33 phenolic acids, 2 C-glycosyl quinochalcones, 6 flavonoid O-glycosides, 4 iridoid glycosides, 6 organic acids, 5 amino acids, and 3 nucleosides, were identified or tentatively characterized. In conclusion, the UHPLC-ESI-QTOF/MS method is useful and efficient for in-depth structural elucidation of chemical compounds in complex matrices of herbal medicines such as DHI.

Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: Possible involvement of L-type calcium channels and myocardial contractility.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvianic acid A (SAA), which is the main water-soluble fraction in Radix Salviae Milthiorrhizae, has been widely applied for treating cardiovascular diseases in China. AIM OF THE STUDY: To explore the effects of SAA against myocardial ischemia injury induced by isoproterenol (ISO) in rats and to clarify its underlying myocardial protective mechanisms based on l-type calcium channels and myocardial contractility. MATERIALS AND METHODS: The myocardial ischemia injured rat model was induced by administering ISO (85mg/kg) subcutaneously at evenly spaced intervals throughout the day and night for 2 consecutive days. Serum cardiac biomarkers were analyzed, and heart tissues were isolated and prepared for histopathology assay. The regulatory effects of SAA on the L-type calcium current (ICa-L) in rat ventricular myocytes were observed by the patch clamp technique. The IonOptix Myocam detection system was used to observe the contractility of isolated rat ventricular myocytes. RESULTS: SAA significantly ameliorated changes in heart morphology and electrocardiographic patterns and reduced serum levels of creatine kinase and lactate dehydrogenase in the ISO-induced myocardial ischemia injured rat model. Meanwhile, SAA reduced ICa-L in a concentration-time dependent way with an IC50 of 1.47×10(-5)M, upshifted the current-voltage, activation, and inactivation curves of ICa-L, and significantly inhibited the amplitude of the cell shortening. CONCLUSIONS: These results indicate that SAA exhibits significant cardioprotective effects against the ISO-induced myocardial ischemia injury, potentially through inhibiting ICa-L and decreasing myocardial contractility.

Studies on the active constituents in radix salviae miltiorrhizae and their protective effects on cerebral ischemia reperfusion injury and its mechanism.

BACKGROUND: To extract, purify and identify the active constituents in ethanol extract of Radix Salviae Miltiorrhizae, and to analyze the protective effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats. MATERIALS AND METHODS: Radix Salviae Miltiorrhizae was extracted by ultrasonic extraction, effective parts were extracted by extraction method, compounds were isolated by preparative TLC and preparative HPLC, and structures of compounds were identified by (1)H NMR and (13)C NMR; the effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats were determined by establishing rat model of middle cerebral artery occlusion (MCAO). RESULTS: The experimental data show four compounds were isolated, namely tanshinone IIB, hydroxymethylene tanshinone, salvianolic acid B and 9"'-methyl lithospermate B. Tanshinone IIA could alleviate the symptoms of neurological deficit in rats, the neurological deficit alleviating effect became more obvious with the increase of dose; tanshinone IIA experimental groups could reduce the cerebral infarction size and brain water content in rats, different concentrations of tanshinone IIA could decrease the SOD content and increase the MDA content in the frontal and parietal cortices of ischemic hemisphere in the ischemia reperfusion group, the differences were statistically significant compared with the ischemia reperfusion group. CONCLUSION: Radix Salviae Miltiorrhizae has the protective effects on cerebral ischemia reperfusion injury in rats.

Miltiorins A-D, diterpenes from Radix Salviae miltiorrhizae.

Constituents of the anti-influenza A neuraminidase (NA) active extract from Radix Salviae miltiorrhizae were investigated, resulting in the isolation of four new diterpenes, miltiorins A-D (1-4), together with eight known diterpenes. The structures of 1-4 were assigned by spectroscopic analysis. Miltiorins A-C (1-3) were abietane diterpenes possessing a 2α-acetoxy group and a 12-hydroxy group in common, while miltiorin D (4) was a 11,12-seco-abietane diterpene with a γ-lactone ring. Miltiorin D (4) is the first example of a 11,12-seco-abietane diterpene from natural sources. Anti-NA activities of the isolated diterpenes were evaluated.

Screening of direct thrombin inhibitors from Radix Salviae Miltiorrhizae by a peak fractionation approach.

Thrombin plays a significant role in thromboembolic disease. In this work, a peak fractionation approach combined with an activity assay method was used to screen direct thrombin inhibitors from Radix Salviae Miltiorrhizae (RSM), a famous herbal remedy for the treatment of cardiovascular diseases in China. A total of 91 fractions were collected from the RSM extract, and 19 fractions out of them showed thrombin inhibitory effects with dose-effect relationship. Among them, three compounds were unambiguously identified as 15, 16-dihydrotanshinone I, cryptotanshinone and tanshinone IIA with IC50 values of 29.39, 81.11 and 66.60µM, respectively. The three compounds were reported with direct thrombin inhibition activities for the first time and their ligand-thrombin interactions were explored by a molecular docking research. These results may contribute to explain the medical benefit of RSM for the prevention and treatment of cardiovascular diseases.